Selective CXCR4 binding peptide conjugate and methods for making and using the same

ABSTRACT

The present invention provides a selective CXCR4 binding peptide conjugate (“PC”), and a method for using and producing the same. In particular, the selective CXCR4 binding peptide conjugate of the invention comprises a peptide portion that selectively binds to CXCR4 and a medically useful compound, such as an imaging agent, a diagnostic agent, or a therapeutically or pharmaceutically active compound. In one particular embodiment, the selective CXCR4 binding peptide conjugate (“PC”) is of the formula: 
                         
or a pharmaceutically acceptable salt thereof, wherein a, b, AA 1 , AA 2 , Ar 1 , X 1 , and AA 3  are those defined herein. The peptide conjugate of the invention can be used in a variety of medical applications including, but not limited to, a targeted drug delivery or imaging a patient or diagnosing a patient for a disease or a clinical condition associated with overexpression and/or upregulation of CXCR4, such as cancers, HIV infection, and immune disorders. Compositions, kits and methods are also disclosed herein for such uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/898,434, filed Feb. 17, 2018, which claims the prioritybenefit of U.S. Provisional Application No. 62/554,354, filed Sep. 5,2017, all of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a selective CXCR4 binding peptideconjugate (“PC”), and a method for using and producing the same. Inparticular, the selective CXCR4 binding peptide conjugate of theinvention comprises a peptide portion that selectively binds to CXCR4and a medically useful compound, such as an imaging agent, a diagnosticagent, or a therapeutically or pharmaceutically active compound.

BACKGROUND OF THE INVENTION

CXCL12 (also called stromal cell-derived factor-1 or SDF-1) and CXCR4, achemokine and chemokine receptor pair play important roles inhematopoiesis, multiple stages of tumorigenesis, and embryonicdevelopment. For example, activation of CXCR4 by CXCL12 has shown todirect leukocyte chemotaxis in the immune system in response toinflammation and progenitor cell migration during embryologicdevelopment. Activation of CXCR4 by CXCL12 has also been shown tomediate signaling pathway that is involved in breast cancer metastasisand memory T cell migration.

CXCR4, a G-protein-coupled receptor also known as fusin or CD184(cluster of differentiation 184), is constitutively- or over-expressedin a wide variety of human cancers, promoting local tumor cellproliferation, survival and angiogenesis. It has also been reported thatCXCR4 is a co-receptor for HIV entry and infection of host cells and hasbeen evaluated as a potential HIV therapy.

Reports have confirmed that CXCR4 is overexpressed in numerous humancancers. CXCR4 antagonism has been shown to disrupt tumor-stromalinteractions, sensitize cancer cells to cytotoxic drugs, and reducetumor growth and metastatic burden. Hence, CXCR4 is a target not onlyfor potential therapeutic intervention of cancer treatment, but also fornoninvasive monitoring of disease progression, therapeutic guidance, andother diagnostic purposes. Some have even suggested that binding andinteracting with CXCR4 as a potential way of targeted drug delivery.

Thus, it is believed that compounds having a moiety that can selectivelybind CXCR4 (i.e., CXCR4 selective binding conjugate) can have a widevariety uses including, but not limited to, treating a wide array ofclinical conditions associated with activation or over-expression ofCXCR4, diagnosing a patient, and medical imaging. Moreover, by labellinga moiety that can selectively bind to CXCR4, one can study the variousmode of drug delivery, drug interaction, diagnosis, in vivo imaging ofcells affected by over-expression of CXCR4, etc. It would be moreuseful, if such a labelled compound do not significantly alter theinteraction with CXCR4.

Accordingly, there is a need for conjugates that can selectively bind toCXCR4 having a medically useful compound. In addition, there is a needfor a labelled conjugate that does not significantly alter the physicalproperties, such as a binding constant, drug activity, itsthree-dimensional structure, etc.

SUMMARY OF THE INVENTION

The present invention provides a selective CXCR4 binding peptideconjugate (“PC”), and a method for using and producing the same. Inparticular, the selective CXCR4 binding peptide conjugate of theinvention comprises a peptide portion that selectively binds to CXCR4and a medically useful compound, such as an imaging agent, a diagnosticagent, or a therapeutically or pharmaceutically active compound. Thepeptidyl portion of the compounds can optionally be isotopicallylabelled. In this manner, one can readily trace, image, or study thecompounds in vivo. The peptide or peptidyl portion is linked to themedically useful compound via a polymeric linker. The peptide conjugateof the invention can be used in a variety of medical applicationsincluding, but not limited to, a targeted drug delivery, imaging apatient, studying the medically useful compound in vivo (e.g.,interaction with CXCR4, etc.), diagnosing a patient for a disease or aclinical condition associated with overexpression and/or upregulation ofCXCR4, such as cancers, HIV infection, and immune disorders.Compositions, kits and methods are also disclosed herein for such uses.

In one particular aspect of the invention provides a selective CXCR4binding peptide conjugate (“PC”) of the formula:

or a pharmaceutically acceptable salt thereof,wherein:

a is 0 or 1;

b is an integer from 1 to 4;

AA¹ along with the sulfur atom that is attached thereto is3-mercaptopropionic acid, optionally substituted cysteine, optionallysubstituted homocysteine, or optionally substituted penicillamine;

AA² along with the sulfur atom that is attached thereto is cysteine orhomocysteine;

Ar¹ is an optionally substituted aryl;

X¹ is Arg, Dap, Dab, Orn, Lys, Dap(iPr), Dab(iPr), Orn(iPr), orLys(iPr);

each of AA³ is independently Gly, Phe, 2Nal, 1Nal, Arg, Dap, Dab, Orn,Lys, Dap(iPr), Dab(iPr), Orn(iPr), Lys(iPr);

and wherein at least one of AA¹, X¹, AA², or AA³ comprises a moiety ofthe formula:-L¹-Q,

wherein

-   -   L¹ is a polymeric linker having a functional group for linking        Q, wherein L¹ comprises from about 2 to about 20 monomers or        copolymers;    -   Q is absent, AA⁴, or a moiety of the formula:        -[AA⁴-Y¹-L²]_(c)-Y²-Z,    -   wherein        -   is 0 or 1;        -   AA⁴ is an amino acid or a derivative thereof;        -   Y¹ is a side-chain function group of amino acid AA⁴;        -   L² is a non-polymeric linker;        -   Z a medically useful compound; and        -   Y² is a functional group of said medically useful compound            or a functional group of L²;            and wherein one or more of AA¹, AA², X¹, AA³, and AA⁴ are            optionally isotopically labelled.

The medically useful compounds that can be used in the inventioninclude, but are not limited to, compounds that can be used indiagnosis, treatment, or assaying various clinical conditions. Exemplarymedically useful compounds include, but are not limited, imaging agents,contrast agents, and therapeutic agents (e.g., drugs).

In some embodiments, the selective CXCR4 binding peptide conjugate ofthe invention is of the formula:

wherein a, b, AA¹, X¹, AA², AA³, L¹, Q, and Ar¹ are those definedherein. Throughout this disclosure, the terms “those defined herein” and“those defined above” when referring to a variable incorporates byreference the broad definition of the variable as well as any narrowerdefinitions including preferred, more preferred and most preferreddefinitions, if any.

In some embodiment, Q is absent from compounds of formulas disclosedherein. In this manner, at least one of AA¹, X¹, AA², or AA³ comprises amoiety of the formula -L¹. Such a compound can be used as a startingmaterial for attaching or linking AA⁴, -AA⁴-Y¹-L², or a moiety of theformula: -[AA⁴-Y¹-L²]_(c)-Y²-Z.

Still in other embodiments, Q is AA⁴. Such a compound can be used forattaching or linking a moiety of the formula -Y¹-L²-Y²-Z.

Yet in other embodiments, Q is a moiety of the formula:-[AA⁴-Y¹-L²]_(x)-Y²-Z. Thus, the invention provides both startingmaterials for attaching a medically useful compound as well as selectiveCXCR4 binding peptide conjugates that include at least one medicallyuseful compound.

In other embodiments, a is 0. Still in other embodiments, a is 1.

Yet still in other embodiments, at least one of X¹ and AA³ isisotopically labelled. In other embodiments, both X¹ and AA³ areisotopically labelled. Exemplary isotopic labels include, but are notlimited to, deuterium, tritium, ¹³C, ¹⁴C, ¹⁸O, and a combinationthereof. In one particular embodiment, isotopic label comprisesdeuterium or tritium. Typically, the isotopically labelled X¹ and/or AA³comprises deuterium, tritium, ¹³C, ¹⁴C, ¹⁸O, or a combination thereof.In one particular embodiment, at least one of X¹ and AA³ is isotopiclabelled with deuterium or tritium. In one specific embodiment, X¹ andAA³ are isotopically labelled Lys(iPr). More specifically, in oneembodiment, X¹ and AA³ are both Lys(deuterated iPr).

Still yet in other embodiments, the medically useful compound is atherapeutic agent. In some embodiments, the therapeutic agent is ananticancer agent. Exemplary anticancer agents that can be used in theinvention include, but are not limited to, paclitaxel, Altretamine,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,Dacarbazine, Diethylstilbestrol, Eribulin, Ethinyl, estradiol,Etoposide, Mitomycin, Mitotane, Mitoxantrone, Pentastatin, Pipobroman,Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen,Teniposide, Vinblastine, and Vincristine. In general, any anticanceragent that has a functional group suitable for linkage can be used. Suchanticancer agents will be readily apparent to one skilled in the arthaving read the present disclosure.

In other embodiments, the selective CXCR4 binding peptide conjugate ofthe invention of the formula:

wherein a, b, AA¹, X¹, AA², AA³, AA⁴, Y¹, Y², L², Z, and Ar¹ are thosedefined herein; and L^(1a) is a polymeric linker comprising from about 2to about 20 monomers or copolymers.

Yet in some embodiments, L² is a non-polymeric linker of the formula:—CH₂—C(═O)—NH—CH₂—, —CH₂—NH—C(═O)—CH₂—, —(CH₂)_(n)—C(═O)—NH—CH₂—,—(CH₂)_(n)—NH—C(═O)—CH₂—, or —(CH₂)_(n)—, wherein n= is an integer from1 to 6.

Still in other embodiments, AA¹ along with the sulfur atom that isattached thereto is optionally substituted cysteine or optionallysubstituted homocysteine. For example, the α-amino group of AA¹ can besubstituted with an acyl group (e.g., acetyl group, etc.), alkyl group(e.g., methyl, dimethyl, ethyl, dimethyl, propyl, dipropyl, iso-propyl,di(iso-propyl), etc.), haloalkyl (e.g., trifluoromethyl,ditrifluoromethyl, etc.). Other suitable substituents for AA¹ will bereadily apparent to one skilled in the art having read the presentdisclosure.

Similar to AA¹, AA² along with the sulfur atom that is attached theretocan also be an optionally substituted cysteine or optionally substitutedhomocysteine.

In one particular embodiment, X¹ and/or at least one of AA³ is Lys(iPr)or Lys(deuterated-iPr). When deuterated (e.g., “deuterated-iPr”),iso-propyl group (“iPr”) can have one or more hydrogen atom that isreplaced with a deuterium. In some embodiments, at least 2, typically atleast 3, often at least 4, more often at least 5 and most often at least6 hydrogen atoms of iPr are replaced with deuterium atoms. Throughoutthis disclosure, a short hand notation for amino acids AA(X) refers toamino acid AA that is substituted with “X” on the side chain functionalgroup. For example, Lys(iPr) refers to lysine that is substituted withiso-propyl group on the amine functional group of lysine's side chain.

Yet other embodiments of the invention, Ar¹ is phenyl. In one particularembodiment, Ar¹ is of the formula:

Still in another particular embodiment of the invention, b is 1. Withinthis embodiment, in some instances, b is 1 and AA³ is Lys(iPr) orLys(deuterated-iPr).

In another embodiment of the invention, L¹ is a polymeric linker of theformula: —NH—(CH₂)₂—[O—CH₂—CH₂]_(n)—O—(CH₂)₂—C(═O)—, wherein n is aninteger from 2 to 20, typically, from 2 to 15, often from 2 to 10, stillmore often from 2 to 8, yet more often from 2 to 6, and most often 3 to6. In some instances, —NH— is attached to the carbonyl carbon of aminoacid AA³. Still in other instances, —C(═O)— is attached to α-aminofunctional group of amino acid AA⁴.

In one particular embodiment, AA⁴ is cysteine, homocysteine, orpenicillin amine.

Still in other embodiments, at least one of AA¹, X¹, AA², or AA³ aminoacid residue is a (D)-isomer.

Yet in other embodiments, a is 0.

In yet other embodiments, AA¹ is homocysteine.

Exemplary selective CXCR4 binding peptide conjugates of the inventioninclude, but are not limited to, Compound of Formula A, Compound ofFormula B, Compound of Formula C, Compound of Formula D, Compound ofFormula E, Compound of Formula F, Compound of Formula G, Compound ofFormula H, Compound of Formula I, Compound of Formula J, Compound ofFormula K, Compound of Formula L, Compound of Formula M, Compound ofFormula N, Compound of Formula O, and Compound of Formula P, as well asthe corresponding deuterium labelled compounds (i.e., -D12) and tritiumlabelled compounds (i.e., -T12).

Another aspect of the invention provides a method for treating a subjectsuffering from a cancer, said method comprising administering to thesubject a therapeutically effective amount of a selective CXCR4 bindingpeptide conjugate disclosed herein, wherein said medically usefulcompound is an anticancer agent. Exemplary anticancer agents usedmethods of the invention include, but are not limited to, paclitaxel,Altretamine, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,Dacarbazine, Diethylstilbestrol, Eribulin, Ethinyl, estradiol,Etoposide, Mitomycin, Mitotane, Mitoxantrone, Pentastatin, Pipobroman,Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen,Teniposide, Vinblastine, and Vincristine.

In some embodiments, methods of the invention provide treatment ofcancer selected from the group consisting of breast cancer, lung cancer,pancreatic cancer, kidney cancer, brain cancer, blood cancer, leukemia,prostate cancer, ovarian cancer, and bladder cancer.

DETAILED DESCRIPTION OF THE INVENTION

CXCR4 plays an important role in immune and inflammatory responses invarious diseases and disorders, including cancer, viral infections, aswell as autoimmune pathologies such as rheumatoid arthritis. The presentinvention is based at least in part on reducing or preventingoverexpression or activation of CXCR4 to treat, diagnose or image aclinical condition associated with CXCR4 overexpression and/oractivation. As used herein, the term “overexpression and/or activation”refers to expression of a gene above its normal (i.e., control) orbaseline level and/or activation of CXCR4 above its normal, control orbaseline level, respectively.

The terms “normal,” “baseline level” and “control level” are usedinterchangeably herein and refer to expression and/or activity level ofCXCR4 in subject(s) that do not have a disease or a clinical conditionassociated with overexpression and/or activation of CXCR4, such as thosedisclosed herein. In some embodiments, the baseline level can be anormal level, meaning the level in a sample from a normal subject thatdo not have a clinical condition associated with overexpression and/oractivation (or activity) of CXCR4. This allows a determination based onthe baseline level of CXCR4 expression or its biological activity, i.e.,whether a sample to be tested or evaluated for disease or a clinicalcondition has a measurable increase, decrease, or substantially nochange in CXCR4 expression or activation as compared to the baselinelevel.

It should be appreciated that the overexpression and/or activation ofCXCR4 can also be determined by comparing the sample result with apositive control. The term “positive control” as used herein refers to alevel of CXCR4 expression and/or activation (or activity) established ina sample from a subject or from a population of individuals, where thesample was believed, based on data from that sample, to have a diseaseor a clinical condition associated with overexpression and/or activationof CXCR4 (e.g., cancer, autoimmune disease such as rheumatoid arthritisand viral infection, such as HIV infection).

In other embodiments, the baseline level can be established from aprevious sample from the subject being tested, so that the diseaseprogression or regression of the subject can be monitored over timeand/or the efficacy of treatment can be evaluated.

Some aspects of the invention provide compounds that have a highaffinity toward CXCR4 that is attached to a diagnostic agent, atherapeutic agent or an imaging agent, through a linker. Such compoundsinclude a CXCR4 binding moiety and a medically active component (orsimply “an active compound”). As used herein, the term “medically activecompound” refers to a compound that is a therapeutically active, or canbe used in diagnostic or imaging, or any other uses associated withtreatment, diagnostic, imaging, analysis or other uses in clinicalapplications. The invention also provides methods for using the same,e.g., in targeted delivery of therapeutics to treat clinical conditionsmanifested by or associated with overexpression and/or activation ofCXCR4, imaging (e.g., in vivo or in vitro) cells associated with CRCR4,identifying cells overexpressing or cells having activated CXCR4, etc.As used herein, the term “high affinity” or “selective” means thecompound or the moiety that binds to CXCR4 has a binding constant(K_(b)) of about 10 nM or less, typically about 3 nM or less, and often1 nM or less. Alternatively, the term “high affinity” or “selective”means the compound or the moiety that binds to CXCR4 has 50% bindinginhibition concentration (IC₅₀) of about 30 nM or less, typically about10 nM or less and often about 3 nM or less. Methods for determiningbinding constant and IC₅₀ are well known to one skilled in the art. See,for example, commonly assigned U.S. provisional patent application Nos.62/384,132, filed Sep. 6, 2016, and 62/505,064, filed May 11, 2017, andcommonly assigned PCT patent application no. PCT/US17/50106, filed Sep.5, 2017, all of which are incorporated herein by reference in theirentirety. In particular, the values K_(b) and IC₅₀ are determined usingthe CXCR4/¹²⁵I-SDF-1α binding assay described in the above referencedprovisional patent applications.

When referring to a numerical value, the term “about” and“approximately” are used interchangeably herein and refer to beingwithin an acceptable error range for the particular value as determinedby one of ordinary skill in the art. Such a value determination willdepend at least in part on how the value is measured or determined,e.g., the limitations of the measurement system, i.e., the degree ofprecision required for a particular purpose. For example, the term“about” can mean within 1 or more than 1 standard deviation, per thepractice in the art. Alternatively, the term “about” when referring to anumerical value can mean ±20%, typically ±10%, often ±5% and more often±1% of the numerical value. In general, however, where particular valuesare described in the application and claims, unless otherwise stated,the term “about” means within an acceptable error range for theparticular value, typically within one standard deviation.

In one particular aspect of the invention, a high affinity or selectiveCXCR4 binding peptide conjugate (“PC”) is of the Formula:

or a pharmaceutically acceptable salt thereof,wherein:

a is 0 or 1;

b is an integer from 1 to 4;

AA¹ along with the sulfur atom that is attached thereto is3-mercaptopropionic acid, optionally substituted cysteine, or optionallysubstituted homocysteine;

AA¹ along with the sulfur atom that is attached thereto is3-mercaptopropionic acid, optionally substituted cysteine, optionallysubstituted homocysteine, or optionally substituted penicillamine;

AA² along with the sulfur atom that is attached thereto is cysteine orhomocysteine;

Ar¹ is an optionally substituted aryl;

X¹ is Arg, Dap, Dab, Orn, Lys, Dap(iPr), Dab(iPr), Orn(iPr), orLys(iPr);

each of AA³ is independently Gly, Phe, 2Nal, 1Nal, Arg, Dap, Dab, Orn,Lys, Dap(iPr), Dab(iPr), Orn(iPr), Lys(iPr);

and wherein at least one of AA¹, X¹, AA², or AA³ comprises a moiety ofthe formula:-L¹-Q,

wherein

-   -   L¹ is a polymeric linker having a functional group for linking        Q, wherein L¹ comprises from about 2 to about 20 monomers or        copolymers;    -   Q is absent, AA⁴, or a moiety of the formula:        -[AA⁴-Y¹-L²]_(c)-Y²-Z,    -   wherein        -   c is 0 or 1;        -   AA⁴ is an amino acid or a derivative thereof;        -   Y¹ is a side-chain function group of amino acid AA⁴;        -   L² is a non-polymeric linker;        -   Z a medically useful compound; and        -   Y² is a functional group of said medically useful compound            or a functional group of L²;            and wherein one or more of AA¹, AA², X¹, AA³, and AA⁴ are            optionally isotopically labelled.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be appreciated that the terms “optionally substituted” whenreferring to an amino acid, means the side chain functional group of theamino acid or the α-amino group of the amino acid may be substituted,for example, by an alkyl (e.g., C₁-C₁₀ alkyl) or a functional groupprotecting group, etc. For example, for lysine or other amino acidshaving a side chain with a nitrogen heteroatom, optionally substitutedor “derivative thereof” includes those substituted with an alkyl, anitrogen protecting group (e.g., acyl containing moieties), and/or ahaloalkyl (e.g., trifluoromethyl, etc.). For cysteine and other sulfurheteroatom containing amino acids (including homocysteine andpenicillamine), the terms “optionally substituted” and “derivativesthereof” can include those that are optionally substituted on the sulfuratom or the α-amine functional group, such as those with alkyl, thiol oramine protecting group, etc. Typically, when referring to AA¹ and AA⁴,the term optionally substituted refers to substitution on the α-aminegroup. Exemplary substitutions on the α-amine group include, but are notlimited to, alkyl group, and amine protecting group, as well as thoseother substituents known to one skilled in the art. “Protecting group”refers to a moiety that when attached to a reactive group in a moleculemasks, reduces or prevents that reactivity. Examples of protectinggroups can be found in T. W. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3^(rd) edition, John Wiley & Sons, New York, 1999,and Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996), which areincorporated herein by reference in their entirety.

AA⁴ is an amino acid or a derivative thereof, where AA⁴ has a side-chainfunctional group Y¹. This presence of the side chain functional groupallows attaching linker L² to the amino acid AA⁴. It should beappreciated that, while the functional group Y¹ when attached to L¹ hasone less hydrogen atom attached thereto. For example, in lysine the sidechain functional group is —NH₂, thus Y¹ when it is not linked to L² is—NH₂ and when it is linked to L², Y¹ is —NH—. Thus, it should beunderstood that the number of hydrogen atom attached to or is present onY¹ is omitted merely for brevity. Furthermore, the side-chain functionalgroup of any amino acid can be readily changed by one skilled in theart. For example, the hydroxy side chain functional group of serine canbe changed to an amine, carboxylic acid, or other functional groups byone skilled in the art using the known reaction procedures. In addition,amine side chain functional group (i.e., —NH₂) of lysine can be changedto azide, —NH—N₃, hydroxy, amide, phosphate, thiol, etc.

In one particular embodiment, X¹ is optionally isotopically. Still inanother embodiment, AA³ is optionally isotopically.

In some embodiments, X¹ and/or AA³ are/is isotopically labelled.Isotopically labelled compound of Formula I can be used inter alia forimaging and/or diagnostic purposes. Exemplary isotopically labelled X¹and/or AA³ include those comprising a positron-emitting radioisotopesuch ³⁴Cl, ⁴⁵Ti, ⁵¹Mn, ⁶¹Cu, ⁶³Zn, ⁶⁸Ga, ¹¹C, ¹³N, ¹⁵O, and ¹⁸F. Whenradioisotope is used, typically such radioisotopes are complexed to X¹and/or AA³ via a complexing or coordinating agent. Suitable complexingor coordinating moieties are well known to one skilled in the art. Otheruseful isotopic labels include, but are not limited to, deuterium (D),tritium (T), ¹³C, ¹⁴C, ¹⁸O, or a combination thereof.

Another example of a useful imaging agent includes a radioactive metalisotope that is coordinated (i.e., chelated) to a chelating group.Particularly useful radioactive metal isotopes include technetium,rhenium, gallium, gadolinium, indium, copper and a combination thereof.Appropriate chelating groups for a particular radioactive metal isotopeare well known to one skilled in the art. For example, ferrocene and itsderivatives, ethylenediaminetetraacetic acid (“EDTA”), its derivatives,a peptidyl moiety Dap-Asp-Cys and its derivatives (see U.S. Pat. No.7,128,893), and others known in the art.

Yet another example of a useful imaging agent includes a contrastingagent. Contrasting agents are widely used, for example, in magneticresonance imaging (MRI). Wide variety of contrasting agents are known toone skilled in the art including gadobenate, gadobutrol, gadodiamide,gadofosveset, gadopentetate, gadoterate, gadoteridol, gadoversetamide,gadoxetate, and iron oxide.

Still another example of a useful imaging agent includes a fluorescentdye, such as fluorenylmethyloxycarbonyl (FMOC) and its derivatives, anAlexaFluor dye, an Oregon Green dye, a fluoresceins, a BODIPY(boron-dipyrromethene) dye, a cyanine dye, a rhodamine dye, a DyLightdye, and Texas Red.

In other embodiments, X¹ and/or AA³ comprises (e.g., attached or linkedto) a diagnostic agent, such as an imaging agent, an isotopic agent, ora radioactive agent. In one particular embodiment, X¹ and/or AA³ isisotopically labelled. In some instances, X¹ and/or AA³ is lysine thatis substituted with isotopically labelled side-chain. As used herein,the term “side-chain” or “side chain” when referring to an amino acid isused to describe the “R” group in the following formula: H₂N—CH(R)—CO₂H.Side chain for a given amino acid is well known to one skilled in theart, for example, the side chain or “R” for glycine is H, for serine itis a moiety of the formula —CH₂OH, etc. In other instances, X¹ and/orAA³ is lysine in which the side chain functional group amine issubstituted with an isotopically labelled alkyl group. As used herein,the term “isotopically labelled alkyl group” means either the carbon orthe hydrogen atom(s) in the alkyl group is replaced with a correspondingisotope such as ¹³C and/or ¹⁴C for carbon atom(s), or deuterium ortritium for hydrogen atom(s). In some instances, the amine nitrogen ofthe lysine side chain functional group is isotopically labelled with¹³N. Still in other embodiments, the carbonyl oxygen of the amino acidis isotopically labelled with ¹⁸O. Yet in other embodiments, one or morecarbon atoms in the amino acid X¹ and/or AA³ is isotopically labelledwith ¹³C or ¹⁴C. Still in other embodiments, α-amino functional group ofamino acid X¹ and/or AA³ is isotopically labelled with ¹⁴N. Yet still inother embodiments, α-amino functional group of amino acid X¹ and/or AA³can be substituted with isotopically labelled group, such asisotopically labelled carbonyl group (e.g., ¹⁸O, ¹³C, ¹⁴C labelledcarbonyl group) or isotopically labelled alkyl group (e.g., D, T, ¹³C,or ¹⁴C).

In one specific embodiment, X¹ and/or AA³ is Lys(iPr) or D-Lys(iPr)where one or more, typically two or more, often three or more, moreoften four or more, still more often five or more and most often atleast six hydrogen atoms on the iPr group is isotopically labelled(i.e., replaced with) with deuterium or tritium.

As can be seen in formula I, in one embodiment the peptidyl group islinked to a medically useful compound of the formula Z by using thefunctional group “Y²” that is present on the medically useful compoundor alternatively that is present on linker L². Such an attachment iswell known and can be readily achieved by one skilled in the art. Forexample, when the functional group Y² is part of the linker L²,medically useful compound Z is modified such that a leaving group orother suitable bond forming agent is present in Z, e.g., if Y² is anamino group of L², it can be used to displace a halide or other suitableleaving groups (e.g., mesylate, tosylate, etc.) or connect to a carbonylgroup that is present in the medically useful compound Z to form anamine or amide linkage, respectively. Similarly, if Y² is a hydroxylgroup, it can be used to displace a halide or other leaving group orconnect to a carbonyl group that is present in the medically usefulcompound to form an ether or an ester, respectively. In a similarmanner, if the function group Y² is present on the medically usefulcompound Z, then Y² can be used to attach linker L². For example, bylinking a hydroxyl or an amino group that is present on the medicallyuseful compound to a carbonyl group that is present on the non-polymericlinker L². In this manner, an ester or an amide bond can be formed,respectively, between the medically useful compound and linker L². Othersuitable functional groups for Y2 will be readily recognized by oneskilled in the art having read to present disclosure.

Still in some embodiments, L² is a non-polymeric linker of the formulaselected from the group consisting of —CH₂—C(═O)—,—(CH₂)_(n)—C(═O)—NH—CH₂—, —(CH₂)_(n)—NH—C(═O)₂—, or —(CH₂)_(n)—, where nis an integer from 1 to 6. In one particular embodiment, L² is—CH₂—C(═O)— and Y² is O.

In formula I, amino acid AA⁴ is an amino acid having a side chain with afunctional group —Y¹. This allows attachment of linker L² to theheteroatom Y¹. Thus, suitable amino acids AA⁴ include, but are notlimited to, serine, cysteine, lysine, arginine, aspartic acid,glutamine, glutamic acid, histidine, proline, threonine, tryptophan,tyrosine, and the like, as well as homologs or derivatives thereof, suchas homocysteine, homoserine. In addition, AA⁴ can also be anon-proteinogenic amino acid, a synthetic amino acid, or an “unnaturalamino acid”, i.e., an amino acid that does not occur naturally but arewell known to one skilled in the art, such as ornithine,1,4-diaminobutyric acid (Dab), 1,3-diaminopropionic acid (Dap),penicillamine, etc. In one particular embodiment, AA⁴ is cysteine,homocysteine, or penicillamine. In such instances, Y¹ is sulfur (e.g.,thiol). In another specific embodiment, AA⁴ is serine, in whichinstances Y¹ is oxygen (e.g., a hydroxyl group).

Unless otherwise stated or context requires otherwise, amino acidsdisclosed herein can be either (D)- or (L)-stereo configuration. Infact, in some embodiments, one or more amino acids are(D)-configuration. In this manner, in vivo stability of compound ofFormula I can be greatly enhanced or increased. Typically, unless statedotherwise amino acids of the invention are proteinogenic amino acids.

Still in other embodiments, L¹ is a polymeric linker comprising fromabout 2 to about 20, typically from about 2 to about 15, often fromabout 2 to about 10, more often from about 2 to 8, and most often fromabout 5 to about 8 monomers or copolymers. Copolymer refers to a linkerhaving two or more different monomeric units. For example, a copolymercan be a polymer that is formed between ethylene glycol propylene glycolunits; between ethylene glycol and vinyl alcohol units; between ethyleneglycol and vinyl acetate units; as well as other copolymers that arewell known to one skilled in the art. It should be appreciated that thetwo end units of L¹ (e.g., one end that is attached to AA³ and the otherend unit that is attached to AA⁴ are functionalized such that it canreadily be linked to AA³ and AA⁴. For example, when L¹ is a polyethyleneglycol (“PEG”), one end of the PEG that is attached to AA³ can befunctionalized as an amine so that it forms an amide bond with AA³. Ifon the other hand, if the end of the PEG that is attached to AA³ ismaintained as a hydroxyl group, then it forms an ester bond with AA³.Similar, if the hydroxyl group of PEG that is attached to AA⁴ isoxidized to a carboxylic acid moiety, then it forms an amide group withthe α-amino functional group of AA⁴. Or when the hydroxyl group of PEGthat is attached to AA⁴ is replaced with a leaving group (e.g.,tosylate, mesylate, halide, etc.), it can form an amino linkage with theα-amino functional group of AA⁴ by, e.g., a displacement reaction. Inthis manner, a wide variety of methods are available for linking L¹ toAA³ and AA⁴. In one specific embodiment, L¹ is a polyethylene glycol inwhich the end attached to AA³ is an amino group, thereby forming anamide bond with AA³. In another embodiment, L¹ is a polyethylene glycolin which the end that is attached to AA⁴ is oxidized to a carboxylicacid or similar functional group, thereby forming an amide linkage withthe α-amino functional group of amino acid AA⁴.

In some embodiments, a is 1. In one particular embodiment, AA³ is lysineor a derivative thereof (e.g., the amino group of the side chain isalkylated, acylated, or substituted with an amine protecting group) thatis optionally isotopically labelled. In some embodiments, the side chainamino functional group is alkylated with an alkyl group optionallyhaving one or more deuterium or tritium in lieu of hydrogen on the alkylgroup. In some embodiments, the side chain amino functional group isacylated that is optionally isotopically labelled, for example,isotopically labelled carbonyl oxygen (e.g., ¹⁸O) and/or isotopicallylabelled carbonyl carbon (e.g., ¹³C or ¹⁴C), etc. In one particularembodiment, AA³ is lysine having an isopropyl group attached to the sidechain amino group. In one specific embodiment, the isopropyl group isoptionally isotopically labelled, e.g., with a plurality of deuteriumand/or tritium atoms.

In some embodiments, a is 0. In this embodiment, compounds have adisulfide bond to afford the ring system.

Still in other embodiments, AA³ is lysine or a derivative thereof (e.g.,the amino group of the side chain is alkylated, acylated, or substitutedwith an amine protecting group) that is optionally isotopicallylabelled. In some embodiments, the side chain amino functional group isalkylated with an alkyl group optionally having one or more deuterium ortritium in lieu of hydrogen on the alkyl group. In some embodiments, theside chain amino functional group is acylated that is optionallyisotopically labelled, for example, isotopically labelled carbonyloxygen (e.g., ¹⁸O) and/or isotopically labelled carbonyl carbon (e.g.,¹³C or ¹⁴C), etc. In one particular embodiment, AA³ is lysine having anisopropyl group attached to the side chain amino group. In one specificembodiment, the isopropyl group is optionally isotopically labelled,e.g., with a plurality of deuterium and/or tritium atoms.

Yet in other embodiments, AA² and the sulfur atom to which it isattached to is cysteine, homocysteine, or penicillamine. Typically, AA²and the sulfur atom to which it is attached to is cysteine orhomocysteine. In one specific embodiment, AA² and the sulfur atom towhich it is attached to is cysteine.

Still in other embodiments, AA¹ and the sulfur atom to which it isattached to is 3-mercaptopropionic acid (3-MPA), cysteine, homocysteine,or penicillamine. Typically, AA¹ and the sulfur atom to which it isattached to is 3-MPA, cysteine or homocysteine. In one specificembodiment, AA¹ and the sulfur atom to which it is attached to iscysteine.

In other embodiments, Ar¹ is phenyl. The —S—CH₂— groups can bepositioned at 1,2-; 1,3-; or 1,4-positions of the phenyl group. In oneparticular embodiment, the —S—CH₂— groups are positioned at1,2-positions, i.e., the phenyl group is 1,2-disubstituted. In otherembodiments, Ar¹ is phenyl having one, two, three, or four, typicallyone, two, or three, often one or two, and most often one substituent(s).Exemplary substituents suitable for Ar¹ include, but are not limited to,halides (e.g., F, Cl, I, or Br), C₁-C₁₀ alkyl (e.g., methyl, ethyl,t-butyl, isopropyl, etc.), C₁-C₁₀ haloalkyl (e.g., —CF₃ etc.), nitro,nitroso, —COnR (where n is 1 or 2 and R is hydrogen or alkyl), cyano,—OR (where R is H, alkyl, carbonyl, etc.), as well as other electrondonating or electron withdrawing groups known to one skilled in the art.

Yet in other embodiments, linker L² comprises a functional group that iscapable of releasing Y²—Z in vivo. In this manner, the moiety Y²—Z isreleased in vivo thereby exerting its therapeutic activity. Suitablefunctional groups that is capable of releasing Y²—Z depends on thenature of the function group on moiety Y² that is linked to the linkerL². For example, when the function group on Z is a hydroxyl group (i.e.,Y² is —OH) or an amino group (Y² is —NH₂), the functional group on L²can be a carboxylate such that an ester bond or an amide bond,respectively, is formed between the medically useful compound and linkerL². If the functional group (i.e., “Y²”) on the medically usefulcompound is a carboxylic acid, the corresponding functional group on L²can be a hydroxyl group or an amino group to form an ester bond or anamide bond, respectively. Other suitable functional groups on L² that iscapable releasing the medically useful compound in vivo are well knownto one skilled in the art including a disulfide bond linkage, an esterlinkage, a thiol-maleimide linkage, and the like.

Still yet in other embodiments, the medically useful compound is atherapeutic agent. Suitable therapeutic agents include those that areknown to one skilled in the art for treatment of cancer, autoimmunedisease (e.g., rheumatoid arthritis), viral infection (e.g., HIVinfection), etc. Exemplary therapeutic agents that are useful incompounds of the invention include, but are not limited to, Altretamine;Asparaginase; Bleomycin; Busulfan; Carboplatin; Carmustine;Chlorambucil; Cisplatin; Cladribine; Cyclophosphamide; Cytarabine;Dacarbazine; Diethylstilbestrol; Eribulin, Ethinyl; estradiol;Etoposide; Mitomycin; Mitotane; Mitoxantrone; Paclitaxel; Pentastatin;Pipobroman; Plicamycin; Prednisone; Procarbazine; Streptozocin;Tamoxifen; Teniposide; Vinblastine; Vincristine, daunorubicin,doxorubicin, docetaxel, irinotecan, monomethyl auristatin E, mertansine,SN-38, tesirine, tubulysin, vinca alkaloids, and an analog or derivativethereof, HIV protease inhibitors, HIV fusion inhibitors, HIV reversetranscriptase inhibitors, HIV integrase inhibitors, HIV entryinhibitors, and therapeutics for autoimmune diseases.

L² can be any biocompatible bifunctional linker such as polyethyleneglycol —(PEG), e.g., in the form of H₂N—CH₂CH₂-(PEG)_(m)-CH₂CH₂—COOH,HOOC—CH₂CH₂-(PEG)m-CH₂CH₂—COOH, or H₂N—CH₂CH₂-(PEG)m-CH₂CH₂—NH₂, naturaland unnatural amino acids or a polyamino acid (PAA), where m is aninteger from 0 to 100, typically 1 to 50, often 1 to 25, and more often1 to 10. Generally, when L² is a polymer (e.g., PEG, PAA), the totalnumber of monomers within the chain is from about 2 (i.e., a monomer) toabout 20, typically from about 2 to about 15, often from about 3 toabout 10, and most often from about 4 to about 6.

Still yet in other embodiments, medically useful compound is adiagnostic or imaging agent, such as a radioactive agent, fluorescentagent, etc. Such imaging agents are well known to one skilled in theart. For example, contrast agents for magnetic resonance imaging agents,ultrasound contrast agents, and radio contrast agents. See, for example,en.wikipedia.org/wiki/Contrast_agent.

Still further, combinations of the various groups described herein canform other embodiments. In this manner, a variety of compounds areembodied within the present invention. By combining various groupsdescribed herein in different manner, compounds such as those of theformulas IA and IB are included within the scope of the invention:

where a, b, AA¹, X¹, AA², AA³, L¹, Q, and Ar¹ are those defined herein,and

where a, b, AA¹, X¹, AA², AA³, AA⁴, Y¹, Y², L¹, L², Z, and Ar¹ are thosedefined herein.

Some specific examples of compounds of the invention include, but arenot limited to, Compound of Formula A, Compound of Formula B, Compoundof Formula C, Compound of Formula D, Compound of Formula E, Compound ofFormula F, Compound of Formula G, Compound of Formula H, Compound ofFormula I, Compound of Formula J, Compound of Formula K, Compound ofFormula L, Compound of Formula M, Compound of Formula N, Compound ofFormula O, and Compound of Formula P, each of which can also be labelledwith deuterium (e.g., Compound of Formulas A-D12, B-D12, C-D12, etc.) ortritium (e.g., Compound of Formulas A-T12, B-T12, C-T12, . . . , P-T12).Synthesis of these compounds are provided in the Examples section.

It should be appreciated that throughout this disclosure combinations ofdifferent embodiments described herein form other preferred embodiments.For example, one particular embodiment is described herein as “a” being0, and another embodiment is described herein where AA¹ is homocysteine.Thus, combination of these two embodiments provides compound of FormulasI, IA, and IB, where a is 0 and AA¹ is homocysteine.

Another aspect of the invention provides a diagnostic kit comprising ahigh affinity CXCR4 selective binding ligand peptide conjugate describedherein where the medically useful compound is a diagnostic agent.

Yet another aspect of the invention provides a composition comprising acompound of Formula I and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier can include a diluent, an excipient,a flavoring agent, an adjuvant, a binder, a stabilizer, coloring agent,or a combination thereof. Generally, “pharmaceutically acceptablecarrier” refers to any excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable, and includes excipient that isacceptable for veterinary use as well as human pharmaceutical use.

The present invention includes pharmaceutical compositions comprising atleast one compound of the invention, or an individual isomer, racemic ornon-racemic mixture of isomers or a pharmaceutically acceptable salt orsolvate thereof, together with at least one pharmaceutically acceptablecarrier, and optionally other therapeutic and/or prophylacticingredients.

In general, the compounds of the invention are administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. Suitable dosageranges are typically 1-500 mg daily, typically 1-100 mg daily, and often1-30 mg daily, depending on numerous factors such as the severity of thedisease to be treated, the age and relative health of the subject, thepotency of the compound used, the route and form of administration, theindication towards which the administration is directed, and thepreferences and experience of the medical practitioner involved. One ofordinary skill in the art of treating such diseases is typically able,without undue experimentation and in reliance upon personal knowledgeand the disclosure of this application, to ascertain a therapeuticallyeffective amount of the compounds of the invention.

Typically, compounds of the invention are administered as pharmaceuticalformulations including those suitable for oral (including buccal andsub-lingual), rectal, nasal, topical, pulmonary, vaginal, or parenteral(including intramuscular, intraarterial, intrathecal, subcutaneous andintravenous) administration or in a form suitable for administration byinhalation or insufflation. Typical manner of administration isgenerally oral using a convenient daily dosage regimen which can beadjusted according to the degree of affliction.

A compound or compounds of the invention, together with one or moreconventional adjuvants, carriers, or diluents, can be placed into theform of pharmaceutical compositions and unit dosages. The pharmaceuticalcompositions and unit dosage forms can be comprised of conventionalingredients in conventional proportions, with or without additionalactive compounds or principles, and the unit dosage forms can containany suitable effective amount of the active ingredient commensurate withthe intended daily dosage range to be employed. The pharmaceuticalcompositions can be employed as solids, such as tablets or filledcapsules, semisolids, powders, sustained release formulations, orliquids such as solutions, suspensions, emulsions, elixirs, or filledcapsules for oral use; or in the form of suppositories for rectal orvaginal administration; or in the form of sterile injectable solutionsfor parenteral use. Formulations containing about one (1) milligram ofactive ingredient or, more broadly, about 0.01 to about one hundred(100) milligrams, per tablet, are accordingly suitable representativeunit dosage forms.

The compounds of the invention can be formulated in a wide variety oforal administration dosage forms. The pharmaceutical compositions anddosage forms can comprise a compound or compounds of the invention orpharmaceutically acceptable salts thereof as the active component. Thepharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances which can also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier generally is a finely divided solid which is a mixture withthe finely divided active component. In tablets, the active componentgenerally is mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from about one (1)to about seventy (70) percent of the active compound. Suitable carriersinclude but are not limited to magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material ascarrier, providing a capsule in which the active component, with orwithout carriers, is surrounded by a carrier, which is in associationwith it. Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be as solid forms suitablefor oral administration.

Other forms suitable for oral administration include liquid formpreparations including emulsions, syrups, elixirs, aqueous solutions,aqueous suspensions, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations. Emulsions canbe prepared in solutions, for example, in aqueous propylene glycolsolutions or may contain emulsifying agents, for example, such aslecithin, sorbitan monooleate, or acacia. Aqueous solutions can beprepared by dissolving the active component in water and adding suitablecolorants, flavors, stabilizers, and thickening agents. Aqueoussuspensions can be prepared by dispersing the finely divided activecomponent in water with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and otherwell known suspending agents. Solid form preparations include solutions,suspensions, and emulsions, and can contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The compounds of the invention can also be formulated for parenteraladministration (e.g., by injection, for example bolus injection orcontinuous infusion) and can be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multidose containerswith an added preservative. The compositions can take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, forexample solutions in aqueous polyethylene glycol. Examples of oily ornonaqueous carriers, diluents, solvents or vehicles include propyleneglycol, polyethylene glycol, vegetable oils (e.g., olive oil), andinjectable organic esters (e.g., ethyl oleate), and can containformulatory agents such as preserving, wetting, emulsifying orsuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution for constitutionbefore use with a suitable vehicle, e.g., sterile, pyrogen-free water.

The compounds of the invention can be formulated for topicaladministration to the epidermis as ointments, creams or lotions, or as atransdermal patch. Ointments and creams can, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions can be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. Formulations suitable for topicaladministration in the mouth include lozenges comprising active agents ina flavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatine andglycerine or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

The compounds of the invention can be formulated for administration assuppositories. A low melting wax, such as a mixture of fatty acidglycerides or cocoa butter is first melted and the active component isdispersed homogeneously, for example, by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and to solidify.

The compounds of the invention can also be formulated for vaginaladministration. Pessaries, tampons, creams, gels, pastes, foams orsprays containing in addition to the active ingredient such carriers asare known in the art to be appropriate.

The compounds of the invention can be formulated for nasaladministration. The solutions or suspensions are applied directly to thenasal cavity by conventional means, for example, with a dropper, pipetteor spray. The formulations can be provided in a single or multidoseform. In the latter case of a dropper or pipette, this can be achievedby the patient administering an appropriate, predetermined volume of thesolution or suspension. In the case of a spray, this can be achieved forexample by means of a metering atomizing spray pump.

The compounds of the invention can be formulated for aerosoladministration, particularly to the respiratory tract and includingintranasal administration. The compound will generally have a smallparticle size for example of the order of five (5) microns or less. Sucha particle size can be obtained by means known in the art, for exampleby micronization. The active ingredient is provided in a pressurizedpack with a suitable propellant such as a chlorofluorocarbon (CFC), forexample, dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, or carbon dioxide or other suitable gas. Theaerosol can conveniently also contain a surfactant such as lecithin. Thedose of drug can be controlled by a metered valve. Alternatively, theactive ingredients can be provided in a form of a dry powder, forexample, a powder mix of the compound in a suitable powder base such aslactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). The powder carrier typicallyforms a gel in the nasal cavity. The powder composition can be presentedin unit dose form, for example, in capsules or cartridges of e.g.,gelatine or blister packs from which the powder can be administered bymeans of an inhaler.

When desired, formulations can be prepared with enteric coatings adaptedfor sustained or controlled release administration of the activeingredient. For example, the compounds of the invention can beformulated in transdermal or subcutaneous drug delivery devices. Thesedelivery systems are advantageous when sustained release of the compoundis necessary or desired and when patient compliance with a treatmentregimen is crucial. Compounds in transdermal delivery systems arefrequently attached to a skin-adhesive solid support. The compound ofinterest can also be combined with a penetration enhancer, e.g., Azone(1-dodecylazacycloheptan-2-one). Sustained release delivery systems canbe inserted subcutaneously into the subdermal layer by surgery orinjection. The subdermal implants encapsulate the compound in a lipidsoluble membrane, e.g., silicone rubber, or a biodegradable polymer,e.g., polylactic acid.

The pharmaceutical preparations are typically in unit dosage forms. Insuch form, the preparation is often subdivided into unit dosescontaining appropriate quantities of the active component. The unitdosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, such as packeted tablets, capsules,and powders in vials or ampoules. Also, the unit dosage form can be acapsule, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form.

Other suitable pharmaceutical carriers and their formulations aredescribed in Remington: The Science and Practice of Pharmacy 1995,edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton,Pa.

When it is possible that, for use in therapy, therapeutically effectiveamounts of a compound of Formula I, as well as pharmaceuticallyacceptable salts thereof, can be administered as the raw chemical, it ispossible to present the active ingredient as a pharmaceuticalcomposition. Accordingly, the disclosure further provides pharmaceuticalcompositions, which include therapeutically effective mounts ofcompounds of Formula I or pharmaceutically acceptable salts thereof or aprodrug thereof, and one or more pharmaceutically acceptable carriers,diluents, or excipients. When applied to a combination, the term refersto combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially, orsimultaneously. The compounds of Formula I and pharmaceuticallyacceptable salts thereof, are as described above. The carrier(s),diluent(s), or excipient(s) must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. In accordance with another aspectof the disclosure there is also provided a process for the preparationof a pharmaceutical formulation including admixing a compound of FormulaI, or a pharmaceutically acceptable salt thereof or a prodrug thereof,with one or more pharmaceutically acceptable carriers, diluents, orexcipients.

When the compositions of this disclosure comprise a combination of acompound of the present disclosure and one or more additionaltherapeutic or prophylactic agent, both the compound and the additionalagent are usually present at dosage levels of between about 10 to 150%,and more typically between about 10 and 80% of the dosage normallyadministered in a monotherapy regimen.

Still another aspect of the invention provides a method for imagingcancer cells in a patient comprising administering to a patient animaging effective amount of a high affinity CXCR4 selective bindingligand peptide conjugate of Formula I, the medically useful compound isan imaging agent, and imaging cancer cells in said patient using animaging apparatus. The imaging apparatus used depends on the nature ofimaging agent of compound of Formula I. For example, if the imagingagent is a positron-emitting radioisotope, then the imaging apparatusused is a PET scan, and when the medically useful compound is acontrasting agent, then the imaging apparatus can be a computedtopography apparatus or an MRI apparatus. When the medically usefulcompound is a radioactive isotope, the imaging apparatus can be an x-raymachine or other similar device.

One particular aspect of the invention provides a method for treatingcancer in a patient. The method comprises administering atherapeutically effective amount of a compound of Formula I (where themedically useful compound is a cancer drug) or a pharmaceuticalcomposition comprising a compound of Formula I (where the medicallyuseful compound is a cancer drug) to a cancer patient.

Another particular aspect of the invention provides a diagnostic or animaging kit comprising a high affinity CXCR4 selective binding ligandpeptide conjugate (PC) of Formula I, where the medically useful compoundis a diagnostic agent or an imaging agent, respectively.

Still another particular aspect of the invention provides a method fortreating a patient suffering from rheumatoid arthritis, pulmonaryfibrosis, HIV infection, or a cancer. The method includes administeringa therapeutically effective amount of a compound of Formula I to apatient in need of treatment thereof. In this method, the medicallyuseful compound of compound of Formula I is a therapeutic agent that canbe used to treat the particular clinical condition to be treated. Someof the cancers that can be treated using compounds of the inventioninclude, but are not limited to, breast cancer, pancreatic cancer,melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin'slymphoma, lung cancer, ovarian cancer, colorectal cancer, multiplemyeloma, glioblastoma multiforme, and chronic lymphocytic leukemia.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

The following abbreviations are used: Ac: acetyl; Boc:tert-butyloxycarbonyl; BOP: (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate; Bz: benzoyl; Bzl: benzyl; Dab:1,4-diaminobutyric acid; Dap: 1,3-diaminopropionic acid; DCC:dicyclohexyl-carbodiimide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; DIEA: dii sopropyl-ethylamine; DMAP:4-(N,N-dimethylamino)pyridine; DMF: N,N-dimethyl formamide; DMSO:dimethyl-sulfoxide; EDT: 1,2-ethane-dithiol; Et: ethyl; Fmoc:9-fluor-enylmethoxy carbonyl; HATU:N-[dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide; HBTU:O-benzo-triazolyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate;HCTU: 1H-benzotriazo-lium1-[bis(dimethylamino)methylene]-5-chloro-3-oxide hexafluorophosphate;HOBt: hydroxybenzotriazole; hCys: homocysteine; iPr: isopropyl; IPA:isopropyl alcohol; Me: methyl; Mmt: 4-mthoxytrityl; Mpa:3-mercaptopropionic acid; 2Nal: 2-naphthylalanine; 1Nal:1-naphthylalanine; NMM: N-methylmorpholine; NMP: N-methyl-pyrrolidone;Orn: ornithine; Pbf:2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl; PBS: phosphatebuffered saline; PyBOP:(benzotriazol-1-yloxy)-tris(pyrrolidino)-phosphoniumhexafluoro-phosphate; PyBrOP: bromotris(pyrrolidino)phosphoniumhexafluorophosphate; tBu: tert-butyl; TFA: trifluoroacetic acid; TFE:trifluroethanol; THF: tetrahydrofuran; TIS: triisopropyl silane; Trt:trityl; mini-PEG6: 6-mer of ethylene glycol; all common amino acids areexpressed as three letter symbols or otherwise specified.

Mass Spectroscopy (MS) Analysis: Preparation of compounds of the presentinvention as described in the following examples is meant to beillustrative rather than limiting. In each of these examples, theobserved molecular weight is reported as a de-convoluted value. Thede-convoluted value is derived from the formula MW (observed)=n(m/z)-n,where m/z represents the charged ion (positive mode) and n is the numberof charges of the specific species. When multiple charged species arepresent in the mass spectrum, the observed molecular weight is reportedas an average.

General Method of Peptide Synthesis, Cyclic Structure Formation, andSalt Exchange: Peptides were synthesized using solid phase peptidesynthesis chemistry known in the art. The cyclic structure of thosepeptides was established, for a disulfide, by using air oxidation, oriodine oxidation in the presence of acidic acid, or for a bisthioetherring, by nucleophilic substitution using a bis(halomethyl) arylcompound, typically using 1.3 equivalents of a bis(bromomethyl) arylcompound, in the presence of a base, such as 15 mM ammonium bicarbonatesolution.

While the present invention illustrates preparation of one particularpeptide linkage, other peptide linkages that are within the scope of thepresent invention can be readily prepared using procedures disclosed in,for example, commonly assigned U.S. patent application Ser. No.15/898,434, filed Feb. 17, 2018, and Ser. No. 15/695,862, filed Sep. 5,2017, both of which are incorporated herein by reference in theirentirety. Furthermore, other peptides linkages that are within the scopeof the present invention can readily be prepared by one skilled in theart having read the present disclosure along with the commonly assignedU.S. patent applications that are incorporated by reference herein.

Paclitaxel Activation—Preparation of 2′-maleimide-paclitaxel:

To a 0° C. solution of 500 mg of paclitaxel (0.6 mmole) in 80 mL of DCMand 0.06 mmole of DMAP (9.2 mg) was added a solution of 1.1 mmole of2-bromoacetic acid (100.3 mg) in 50 mL of DCM followed by 3 eq. of DIC(0.3 mL) under stirring. The reaction mixture was then slowly warmed upto room temperature and the coupling reaction was allowed to proceed at30° C. for one hour under continuous stirring. Crude product of2′-bromoacetyl-paclitaxel was purified and lyophilized beforeconjugation to cyclized CXCR4 antagonist peptide (MW MS: observed975.10; calculated: 974.85).

Purification, Salt Form Conversion, and Final Product Characterization:Final products were purified by reverse phased HPLC and furthercharacterized by analytical HPLC and mass spectroscopy. Peptidespurified from reverse phased HPLC were usually in trifluoroacetic acid(TFA) form. This salt was typically converted to a more pharmaceuticallyfriendly salt form, such as acetic acid or hydrochloric acid salt form.Converting a peptide in TFA salt to a hydrochloric acid salt could beachieved by repeated lyophilization of the peptide in TFA salt in adilute hydrochloric acid solution. For conversion of a peptide in TFAsalt to an acetate salt, typically the following process was used.Strong anion exchange resin (chloride form, substitution 3 mmole/g,water content 50%, using 2 grams of resin per gram of peptide) was firstwashed three times with milli Q water, then three times with 1 N NaOHsolution three times, 5 min/time, and then five times with milli Qwater, 5 min/time. The resin was further washed with 75% ethanol wateruntil the pH reaches about 7.4. This resin was treated with 10% aceticacid solution three times, five minutes each time. The resin was thenwashed with 1% acetic acid solution three times, five minutes each time.The resin was ready for the salt conversion of the purified peptide.

The purified, lyophilized peptide was dissolved in 1% acetic acidsolution and added to the prepared resin described above. The mixturewas agitated or magnetically stirred at room temperature for 1 h. Thesupernatant was separated. The resin was washed three times with 1%acetic acid solution. The supernatant and the washing solution werecombined, filtered through a 0.22 μm membrane and lyophilized, to afforda peptide in acetate salt.

Example 1 Synthesis of Compound A

Peptide chain assembly:Fmoc-Cys(Mmt)-Tyr(tBu)-Lys(iPr,Boc)-(D-Arg(Pbf))-2Nal-Gly-Cys(Mmt)-Lys(iPr,Boc)-(mini-PEG6)-Cys(Trt)-SieberAmide AM Resin (SEQ ID NO:5)

The peptide chain was assembled by standard Fmoc chemistry using Sieberamide AM resin (Xi'an LanXiao Chemical Limited, Xi'an, China). Briefly,40 g of Sieber amide AM resin was swollen in 300 mL of DCM for 2 h andthen washed four times with DMF. Removal of Fmoc was carried out in 250mL of 20% piperidine in DMF for 20 min at room temperature and washedseveral times with DMF. Ninhydrin test was negative. Stepwise chainassembly started with Fmoc-Cys(Trt)-OH from the C-terminal end of thelinear peptide. Three equivalents of protected amino acidFmoc-Cys(Trt)-OH were activated with DIC/HOBt in 80 mL of DMF, andcoupled to the Fmoc-removed Sieber amide AM resin prepared above for 2 hat room temperature. Ninhydrin test was negative. Capping of thenon-reacted amino group was performed for 30 min with 160 mL of amixture of acetic anhydride/DIEA/DCM at a volume ratio of 1:1:4. Thiswas followed by Fmoc removal using 250 mL of 20% piperidine in DMF for20 min. The following residues were coupled sequentially withoutcapping: Fmoc-(mini-PEG6)-OH, Fmoc-Lys(iPr,Boc)-OH, Fmoc-Cys(Mmt)-OH,Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg(Pbf))-OH, Fmoc-Lys(iPr,Boc)-OH,Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Mmt)-OH. After the coupling of lastresidue Fmoc-Cys(Mmt), Fmoc protection was removed again using 250 mL of20% piperidine in DIVIF for 20 min. N-terminal acetylation was carriedout with 160 mL of a mixture of acetic anhydride/DIEA/DMF (1:1:4, v/v/v)for 30 min at room temperature. The resin was then washed with 250 mL ofDIVIF three time and then with 250 mL of DCM three times, then driedunder vacuum to afford 81 grams of peptide resin containing theassembled target linear peptide. The dried resin was divided intoseveral portions and one portion (20 grams) is used in the followingoperation.

Removal of Mmt protection on Cys residues and Cleavage of partialprotected linear peptide from peptide resin: 20 grams of above peptideresin was swelled in 300 mL of DCM for 30 min. Total 1000 mL of acleavage/deprotection cocktail (TFA/H₂O/EDT/TIS/DCM, 6:3:1.5:3:86.5,v/v) was added to above swelled resin to remove Mmt protection of Cysside chain. The mixture was stirred for 20 min at room temperature. Thisdeprotection and cleavage procedure was repeated once using another 1000mL of the cleavage/deprotection cocktail above at room temperature for20 min. After removal of the solid resin by filtration, the cleavagesolutions containing the partially deprotected linear peptide werecombined and concentrated under vacuum using a rotary evaporator. Theresidues (crude peptide) were then lyophilized to afford 9.5 grams ofpartially deprotected crude linear peptide.

Cyclization: To a solution of 2.2 grams of above crude linear peptide in110 mL of DMF was added 20 mL of MeCN containing 368 mg of1,2-bis(bromomethyl) benzene. The solutions were mixed well, then 1630mL of MeCN and 440 mL of water were added. The pH of this reactionmixture was adjusted to pH 8˜9 using 1 M of ammonium carbonate solution.The cyclization reaction was allowed to proceed for 1 h at roomtemperature. The reaction was monitored by MS. The reaction mixture wasthen divided into six equal portions and lyophilized.

Side chain deprotection of the cyclized peptide: The lyophilized crudecyclized peptide was deprotected using a cleavage cocktail(TFA/EDT/TIS/H₂O/thioanisole/phenol, per 100 mL of solution contains81.5 mL TFA, 2.5 mL EDT, 1.5 mL TIS, 5.0 mL H₂O, 5.0 mL thioanisole, and5.0 grams phenol) at 10 mL each container, for 60 min at 30° C. To thecleavage mixture was added 4 volumes of cold ethyl ether. The crudepeptide precipitates were separated by centrifugation at 3000 rpm for 2min. The crude peptide precipitates were washed three times with ethylether. The crude peptide was purified to a purity >90% on preparativeHPLC, and lyophilized (MW MS: observed 1719.55; calculated: 1720.18).

Preparation of PDC-CXCR4 peptide paclitaxel drug Conjugate: Aboveprepared 452 mg of 2′-bromoacetyl-paclitaxel and 905 mg of cyclizedCXCR4 peptide were dissolved in 100 mL of MeCN/water (1:1, v/v). Thesolution was adjusted to pH 7˜7.5 using 0.5M of ammonium bicarbonate.The conjugation reaction was completed in about half an hour asconfirmed by MS at room temperature. The final product was purifiedusing a reverse-phased preparative column Daisogel (50×250 mm, 8 μm);mobile phases—Solvent A: 0.1% TFA water; Solvent B: 0.1% TFAacetonitrile. Fractions containing the target product MB1707 werecombined and lyophilized (a TFA salt). Salt exchange as described aboveafforded a peptide in acetate salt. Analytical HPLC purity of the finalpeptide product is 99.61%; MW cal.: 2614.12 Da; MW obs.: 2613.30 Da.

Example 2 Synthesis of Isotopically Labelled Compound A-D12

Peptide chain assembly:Fmoc-Cys(Mmt)-Tyr(tBu)-Lys(Boc)-(D-Arg(Pbf))-2Nal-Gly-Cys(Mmt)-Lys(Boc)-(mini-PEG6)-Cys(Trt)-SieberAmide AM Resin (SEQ ID NO:7).

The peptide chain was assembled by standard Fmoc chemistry using Sieberamide AM resin (Xi'an LanXiao Chemical Limited, Xi'an, China). Briefly,1.5 grams of Sieber amide AM resin was swollen in 30 mL of DCM for 2 hand then washed four times with 25 mL of DMF. Removal of Fmoc wascarried out in 25 mL of 20% piperidine in DMF for 20 min at roomtemperature and washed several times with DMF. Ninhydrin test wasnegative. Stepwise chain assembly started with Fmoc-Cys(Trt)-OH from theC-terminal end of the linear peptide. Three equivalents of protectedamino acid Fmoc-Cys(Trt)-OH (702 mg) were activated with DIC (1 mL)/HOBt(243 mg) in 5 mL of DMF, and coupled to the Fmoc-removed Sieber amide AMresin prepared above in a temperature controlled oven at 30° C.overnight. The reaction mixture was drained and the resin was washedseveral times with 25 mL of DMF. Capping of the non-reacted amino groupwas performed for 30 min with 16 mL of a mixture of aceticanhydride/DIEA/DCM at a volume ratio of 1:1:4. Ninhydrin test wasnegative. This was followed by Fmoc removal using 25 mL of 20%piperidine in DMF for 20 min.

The following residues were coupled sequentially without the cappingstep: Fmoc-(mini-PEG6)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Mmt)-OH,Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg(Pbf))-OH, Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)-OH, and Fmoc-Cys(Mmt)-OH. After the coupling of lastresidue Fmoc-Cys(Mmt), Fmoc protection was removed again using 25 mL of20% piperidine in DMF for 20 min. N-terminal acetylation was carried outwith 16 mL of a mixture of acetic anhydride/DIEA/DMF (1:1:4, v/v/v) for30 min at room temperature. The peptide resin was then washed with 25 mLof DMF three time and followed by 25 mL of DCM three times, then thepeptide resin dried under vacuum to afford 2.8 grams of peptide resincontaining the assembled target linear peptide.

Removal of Mmt protection on Cys residues and Cleavage of partialprotected linear peptide from peptide resin: 2.8 grams of above peptideresin was swelled in 30 mL of DCM for 30 min. 150 mL of acleavage/deprotection cocktail (TFA/H₂O/EDT/TIS/DCM, 6:3:1.5:3:86.5,v/v) was added to above swelled resin to remove Mmt protection of Cysside chain, 20 min at room temperature. The supernatant of the cleavagemixture was drained into a flask containing 150 mL of water. Thisdeprotection and cleavage procedure was repeated once using another 150mL of the cleavage/deprotection cocktail above at room temperature for20 min. After removal of the solid resin by filtration, the cleavagesolutions containing the partially deprotected linear peptide werecombined and concentrated under vacuum using a rotary evaporator. Theresidues (crude peptide) were then lyophilized to afford 1.2 grams ofpartially deprotected crude linear peptide.

Cyclization: To a solution of 1.2 grams of above crude linear peptide in60 mL of DMF was added 10 mL of MeCN containing 200 mg of1,2-bis(bromomethyl) benzene. The solutions were mixed well, then 890 mLof MeCN and 240 mL of water were added. The pH of this reaction mixturewas adjusted to pH 8˜9 using 1 M of ammonium carbonate solution. Thecyclization reaction was allowed to proceed for 1 h at room temperatureunder magnetic stirring. The completion of cyclization was confirmed byMS. The reaction mixture was then divided into three equal portions andlyophilized in a flask.

Side chain deprotection of cyclized peptide: To each of the lyophilizedcrude cyclized peptide flask was added 10 mL of a cleavage cocktailcomposed of TFA/EDT/TIS/H₂O/thioanisole/phenol (per 100 mL of solutioncontains 81.5 mL TFA, 2.5 mL EDT, 1.5 mL TIS, 5.0 mL H₂O, 5.0 mLthioanisole, and 5.0 grams phenol). The cleavage reaction was allowed toproceed for 60 min at 30° C. To the cleavage mixture was added 4 volumesof cold ethyl ether. The crude peptide precipitates were separated bycentrifugation at 3000 rpm for 2 min. The crude peptide precipitateswere washed three times with icy cold ethyl ether. The crude peptide wasdried under vacuum and then used for lysine side chain modification asfollowing without further purification.

Lysine side chain modification using acetone-D6: 125 mg of crude productfrom above was dissolved in 12.5 mL of a solution composed of aceticacid:acetone-D6:ethanol (2:2:8.5, v/v), then 324 mg of sodiumcyanoboronhydride (NaBH₃CN) was added under stirring. The reductiveamination was allowed to proceed at 30° C. for 2 h. The reaction mixturewas diluted with water and loaded onto a preparative HPLC column. Theisotopically labeled cyclic peptide was purified to a purity >90% andlyophilized.

Preparation of PDC-CXCR4 Peptide Paclitaxel Drug Conjugate:

Above prepared 452 mg of 2′-bromoacetyl-paclitaxel and 905 mg ofcyclized CXCR4 peptide were dissolved in 100 mL of MeCN/water (1:1,v/v). The solution was adjusted to pH 7˜7.5 using 0.5M of ammoniumbicarbonate. The conjugation reaction was completed in about half anhour as confirmed by MS at room temperature. The final product waspurified using a reverse-phased preparative column Daisogel (50×250 mm,8 μm); mobile phases—Solvent A: 0.1% TFA water; Solvent B: 0.1% TFAacetonitrile. Fractions containing the target product MB1707-D12 werecombined and lyophilized (a TFA salt). Salt exchange as described aboveafforded a peptide in acetate salt. Analytical HPLC purity of the finalpeptide product is 99.01%; MW cal.: 2626.20 Da; MW obs.: 2623.35 Da.

Example 3 Synthesis ofCyclic[Ac-HCys-Tyr-Lys(iPr)-d-Arg-2Nal-Gly-Cys]-Lys(iPr)-PEG₆-COOH, adisulfide ring hCys1-Cys7

The sequence hCys(Trt)-Tyr(tBu)-Lys(iPr,Boc)-(D-Arg(Pbf))-2Nal-Gly-Cys(Trt)-Lys(iPr, Boc)-PEG6 (SEQ ID NO:11)was assembled by standard Fmoc chemistry using 2-chlorotrityl chlorideresin. Briefly, 4.0 grams of the resin was swollen in DCM for 2 h,washed four times with DMF and then once with DCM. The loading of thefirst residue Fmoc-PEG6 was carried out in DCM using four equivalents ofamino acid activated with five equivalents of DIEA. The coupling at roomtemperature for 1.5 h was followed by capping of the unreactedsubstitution sites with methanol/DIEA (1:1, v/v, 24 mL) for 30 min.Removal of Fmoc protection was carried out in 20% piperidine in DMF for20 min at room temperature and washed several times with DMF. Ninhydrintest was negative. Stepwise chain assembly of the linear peptide usingstandard Fmoc chemistry was continued consequentially through a cycle ofdeprotection, activation and coupling.

After the coupling of last residue Fmoc-hCys(Trt)-OH, Fmoc protectionwas removed again using 20% piperidine in DMF for 20 min. N-terminalacetylation was carried out with 5 mL of a mixture of aceticanhydride/DIEA/DMF (1:1:4, v/v/v) for 30 min at room temperature. Theresin was then washed with DMF three time and then with DCM twice, driedunder vacuum.

The crude linear peptide cleavage and side chain protections werecarried out using 10 mL of a cleavage cocktail ofTFA/EDT/TIS/H₂O/thioanisole/phenol per gram of crude peptide resin (per100 mL of solution contains 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mLH₂O, 5.0 mL thioanisole, and 5.0 grams phenol) for 70 min at roomtemperature. Then to the cleavage mixture was added eight volumes ofmethyl t-butyl ether. The crude peptide precipitates were separated bycentrifugation at 3000 rpm for 3 min. The crude peptide precipitateswere washed three times with methyl t-butyl ether. The crude peptide wasthen dissolved in an aqueous acetonitrile and lyophilized.

The crude lyophilized product was used directly in the cyclizationreaction. The lyophilized crude peptide was dissolved in 20% acetic acidat 0.5 mg/mL. Under stirring, add 0.03% mole/L iodine solution until thepeptide solution turned light yellow. The solution was protected fromvisible light during the cyclization. Cyclization was completed within0.5 h as monitored by mass spectroscopy. The cyclized final product waspurified using a reverse-phased preparative column Daisogel (50×250 mm,8 μm) with mobile phases—A: 0.1% TFA water; B: 0.1% TFA acetonitrile.Fractions containing the target product were combined and lyophilized (aTFA salt).

Example 4 Synthesis ofcyclic[Ac-hCys-Tyr-Lys(iPr)-d-Arg-2Nal-Gly-Cys]-Lys(iPr)-PEG₆-Lys, aDisulfide Ring hCys1-Cys7

The sequencehCys(Trt)-Tyr(tBu)-Lys(iPr,Boc)-(D-Arg(Pbf))-2Nal-Gly-Cys(Trt)-Lys(iPr,Boc)-PEG6-Lys(Boc)(SEQ ID NO:13) was assembled manually by standard Fmoc chemistry usingRink AM resin. Briefly, 3.6 grams of Rink AM resin was swollen in DCMfor 14 h and then washed four times with DMF. Removal of Fmoc wascarried out in 20% piperidine in DMF for 20 min at room temperature andwashed several times with DMF. Ninhydrin test was negative. Stepwisechain assembly started from the C-terminal end of the linear peptide andwas accomplished in nine major steps. In step 1, three equivalents ofprotected amino acid Fmoc-Lys(Boc) were activated with DIC/HOBt in DMF,and coupled to the Fmoc-removed Rink AM resin above for 2 h at roomtemperature followed by Fmoc removal using 20% piperidine in DMF for 20min. In step 2, three equivalents of Fmoc-PEG6 acid were activated withDCC/HOBt in DMF and coupled to the deprotected resin from step 1.Appropriate steps were continued using Fmoc-protected amino acids,respectively, until the coupling of Fmoc-hCys(Trt)-OH.

After the coupling of last residue Fmoc-hCys(Trt)-OH, Fmoc protectionwas removed again using 20% piperidine in DMF for 20 min. N-terminalacetylation was carried out with 5 mL of a mixture of aceticanhydride/DIEA/DMF (1:1:4, v/v/v) for 30 min at room temperature. Theresin was then washed with DMF three time and then with DCM twice, driedunder vacuum.

The finished peptide was deprotected and cleaved from the resin using 10mL/gram of resin of a cleavage cocktail ofTFA/EDT/TIS/H₂O/thioanisole/phenol (per 100 mL of solution contains 81.5mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H₂O, 5.0 mL thioanisole, and 5.0grams phenol) for 70 min at room temperature. To the cleavage mixturewas added eight volumes of methyl t-butyl ether. The crude peptideprecipitates were separated by centrifugation at 3000 rpm for 3 min. Thecrude peptide precipitates were washed three times with methyl t-butylether. The crude peptide was then dissolved in aqueous acetonitrile andlyophilized.

The crude lyophilized product was used directly in the cyclizationreaction. The lyophilized crude peptide was dissolved in watercontaining 20% acetic acid at 0.5 mg/mL (500 mg of crude peptide in oneliter). Under stirring, 0.03% mole/L iodine solution was added to thepeptide solution until the solution became pale yellow. Cyclization wascomplete within 0.5 h in dark as monitored by mass spectroscopy. Thecyclized final product was purified using a reverse-phased preparativecolumn Daisogel (50×250 mm, 8 μm); mobile phases—Solvent A: 0.1% TFAwater; Solvent B: 0.1% TFA acetonitrile. Fractions containing the targetproduct were combined and lyophilized (a TFA salt).

Example 5Cyclic[Acetyl-hCys-Tyr-Lys(iPr)-d-Arg-2Nal-Gly-Cys]-Lys(iPr)-mini-PEG₆-Cys-Amide,a Disulfide Ring hCys1-Cys7

The sequencehCys(Mmt)-Tyr(tBu)-Lys(iPr,Boc)-(D-Arg(Pbf))-2Nal-Gly-Cys(Mmt)-Lys(iPr,Boc)-PEG6-Cys(Trt)(SEQ ID NO:15) was assembled by standard Fmoc chemistry using Sieberamide AM resin (Xi'an LanXiao Chemical Limited, Xi'an, China). Briefly,4 g of Sieber amide AM resin was swollen in 30 mL of DCM for 2 h andthen washed four times with DMF. Removal of Fmoc was carried out in 25mL of 20% piperidine in DMF for 20 min at room temperature and washedseveral times with DMF. Ninhydrin test was negative. Stepwise chainassembly started with Fmoc-Cys(Trt)-OH from the C-terminal end of thelinear peptide. Three equivalents of protected amino acidFmoc-Cys(Trt)-OH were activated with DIC/HOBt in 8 mL of DMF, andcoupled to the Fmoc-removed Sieber amide AM resin prepared above for 2 hat room temperature. Ninhydrin test was negative. Capping of thenon-reacted amino group was performed for 30 min with 16 mL of a mixtureof acetic anhydride/DIEA/DCM at a volume ratio of 1:1:4. This wasfollowed by Fmoc removal using 25 mL of 20% piperidine in DMF for 20min. The following residues were coupled sequentially without capping:Fmoc-(mini-PEG6)-OH, Fmoc-Lys(iPr,Boc)-OH, Fmoc-Cys(Mmt)-OH,Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg(Pbf))-OH, Fmoc-Lys(iPr,Boc)-OH,Fmoc-Tyr(tBu)-OH, and Fmoc-hCys(Mmt)-OH. After the coupling of lastresidue Fmoc-hCys(Mmt), Fmoc protection was removed again using 25 mL of20% piperidine in DMF for 20 min. N-terminal acetylation was carried outwith 16 mL of a mixture of acetic anhydride/DIEA/DMF (1:1:4, v/v/v) for30 min at room temperature. The resin was then washed with 25 mL of DMFthree time and then with 25 mL of DCM three times, then dried undervacuum to afford 8 grams of peptide resin containing the assembledtarget linear peptide. The dried resin was used in the followingoperation.

Removal of Mmt protection on Cys residues and Cleavage of partialprotected linear peptide from peptide resin: About 2 grams of abovepeptide resin was swelled in 30 mL of DCM for 30 min. Total 100 mL of acleavage/deprotection cocktail (TFA/H₂O/EDT/TIS/DCM, 6:3:1.5:3:86.5,v/v) was added to above swelled resin to remove Mmt protection of Cysside chain. The mixture was stirred for 20 min at room temperature. Thisdeprotection and cleavage procedure was repeated once using another 100mL of the cleavage/deprotection cocktail above at room temperature for20 min. After removal of the solid resin by filtration, the cleavagesolutions containing the partially deprotected linear peptide werecombined and concentrated under vacuum using a rotary evaporator. Theresidues (crude peptide) were then lyophilized to afford about 1 gram ofpartially deprotected crude linear peptide.

The crude lyophilized product was used directly in the cyclizationreaction. The lyophilized crude peptide was dissolved in watercontaining 20% acetic acid at 0.5 mg/mL (500 mg of crude peptide in oneliter). Under stirring, 0.03% mole/L iodine solution was added to thepeptide solution until the solution became pale yellow. Cyclization wascomplete within 0.5 h in dark as monitored by mass spectroscopy. Thecyclization reaction was allowed to proceed for 1 h at room temperature.The reaction mixture was then divided into several equal portions andlyophilized.

Side chain deprotection of the cyclized peptide: The lyophilized crudecyclized peptide was deprotected using a cleavage cocktail(TFA/EDT/TIS/H₂O/thioanisole/phenol, per 100 mL of solution contains81.5 mL TFA, 2.5 mL EDT, 1.5 mL TIS, 5.0 mL H₂O, 5.0 mL thioanisole, and5.0 grams phenol) at 10 mL each container, for 60 min. To the cleavagemixture was added 4 volumes of cold ethyl ether. The crude peptideprecipitates were separated by centrifugation at 3000 rpm for 2 min. Thecrude peptide precipitates were washed three times with ethyl ether. Thecrude peptide was purified to a purity >90% on preparative HPLC, andlyophilized.

Example 6 Preparation of PDC-CXCR4 Peptide Conjugate Compound F

Above prepared 450 mg of 2′-bromoacetyl-paclitaxel and 905 mg ofcyclized CXCR4 peptide Compound C are dissolved in 100 mL of MeCN/water(1:1, v/v). The solution is adjusted to pH 7˜7.5 using 0.5M of ammoniumbicarbonate. The conjugation reaction is monitored by MS and thereaction completed in about half an hour. The final product is purifiedusing a reverse-phased preparative column Daisogel (50×250 mm, 8 μm);mobile phases—Solvent A: 0.1% TFA water; Solvent B: 0.1% TFAacetonitrile. Fractions containing the target product Compound F arecombined and lyophilized (a TFA salt). Salt exchange as described aboveaffords a peptide in acetate salt.

Example 7 Preparation of Compounds G-P

Compounds GP are prepared in a similar fashion starting with a Rinkamide resin or 2-chlorotrityl resin or a Sieber amide AM resin usingstandard Fmoc solid phase peptide chemistry.

Example 8 Synthesis of Isotopically Labelled Compounds

Using the procedures described in Examples above and using acorresponding deuterated compound B-D12 or a corresponding tritiumlabelled compound (i.e., compound B-T12, for tritium labelled compoundof B, similar to B-D12 but where deuterium are replaced with tritium)deuterated or tritium labelled cyclic peptide moieties are prepared. Useof these deuterium labelled (B-D12) and tritium labelled (B-T12)compounds provide synthesis of corresponding deuterium labelled (“-D12”)and tritium labelled (i.e., “-T12”) compounds A-P, i.e., compoundsA-D12, A-T12, B-D12, B-T12, C-D12, C-T12, . . . P-D12, and P-T12.

Human CXCR4/¹²⁵I-SDF-1α Binding Inhibition Assay: (Performed by EUROFINSCEREP SA, Le Bois l'Eveque, 86600 Celle L'Evescault, France): Humanchemokine receptor CXCR4 expressed in Chem-1 cells were used in modifiedHEPES buffer pH 7.4. A 0.5 μg (Membrane protein may change from lot tolot, the concentration used will be adjusted if necessary), aliquot wasincubated with 0.03 nM [¹²⁵I]SDF-1α for 90 minutes at 25° C.Non-specific binding was estimated in the presence of 30 nM SDF-1α.Membranes were filtered and washed, filters were then counted todetermine [¹²⁵I]SDF-1α specifically bound. Compounds were screenedstarting at 10 μM with 11-point dilutions (Valenzuela-Fernandez A, etal. J Biol Chem. 277(18):15677, 2002). The CXCR4 binding affinity of theantagonist peptides disclosed herein and their drug-conjugates is 1.0 μMor less to be practical for therapeutic use.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A selective CXCR4 binding peptide conjugate(“PC”) of the formula:

or a pharmaceutically acceptable salt thereof, wherein: a is 0 or 1; bis an integer from 1 to 4; AA¹ along with the sulfur atom that isattached thereto is 3-mercaptopropionic acid, optionally substitutedcysteine, optionally substituted homocysteine, or optionally substitutedpenicillamine; AA² along with the sulfur atom that is attached theretois cysteine or homocysteine; Ar¹ is an optionally substituted aryl; X¹is Arg, Dap, Dab, Orn, Lys, Dap(iPr), Dab(iPr), Orn(iPr), or Lys(iPr);each of AA³ is independently Gly, Phe, 2Nal, 1Nal, Arg, Dap, Dab, Orn,Lys, Dap(iPr), Dab(iPr), Orn(iPr), Lys(iPr); and wherein at least one ofAA¹, X¹, AA², or AA³ comprises a moiety of the formula:-L¹-Q, wherein L¹ is a polymeric linker having a functional group forlinking Q, wherein L¹ comprises from about 2 to about 20 monomers orcopolymers; Q is absent, AA⁴, or a moiety of the formula:-[AA⁴-Y¹-L²]_(c)-Y²-Z, wherein c is 0 or 1; AA⁴ is an amino acid or aderivative thereof; Y¹ is a side-chain function group of amino acid AA⁴;L² is a non-polymeric linker; Z a medically useful compound; and Y² is afunctional group of said medically useful compound or a functional groupof L²; and wherein one or more of AA¹, AA², X¹, AA³, and AA⁴ areoptionally isotopically labelled.
 2. The selective CXCR4 binding peptideconjugate according to claim 1, wherein said medically useful compoundcomprises an imaging agent, a contrast agent, or a therapeutic agent. 3.The selective CXCR4 binding peptide conjugate according to claim 1 ofthe formula:

wherein a, b, AA¹, X¹, AA², AA³, L¹, Q, and Ar¹ are as defined inclaim
 1. 4. The selective CXCR4 binding peptide conjugate according toclaim 3, wherein Q is absent.
 5. The selective CXCR4 binding peptideconjugate according to claim 3, wherein Q is AA⁴, or a moiety of theformula: -[AA⁴-Y¹-L²]_(c)-Y²-Z.
 6. The selective CXCR4 binding peptideconjugate according to claim 5, wherein a is
 0. 7. The selective CXCR4binding peptide conjugate according to claim 1, wherein a is
 0. 8. Theselective CXCR4 binding peptide conjugate according to claim 1, whereinAA¹ is homocysteine.
 9. The selective CXCR4 binding peptide conjugateaccording to claim 1, wherein a is
 1. 10. The selective CXCR4 bindingpeptide conjugate according to claim 1, wherein X¹ is isotopicallylabelled.
 11. The selective CXCR4 binding peptide conjugate according toclaim 1, wherein AA³ is isotopically labelled.
 12. The selective CXCR4binding peptide conjugate according to claim 1, wherein at least one ofX¹ and AA³ is isotopic labelled, and wherein said isotopically labelcomprises deuterium, tritium, ¹³C, ¹⁴C, ¹⁸O, or a combination thereof.13. The selective CXCR4 binding peptide conjugate according to claim 12,wherein at least one of X¹ and AA³ is isotopically labeled withdeuterium or tritium.
 14. The selective CXCR4 binding peptide conjugateaccording to claim 1, wherein said medically useful compound is atherapeutic agent.
 15. The selective CXCR4 binding peptide conjugateaccording to claim 14, wherein said therapeutic agent is an anticanceragent.
 16. The selective CXCR4 binding peptide conjugate according toclaim 15, wherein said anticancer agent is selected from the groupconsisting of paclitaxel, Altretamine, Asparaginase, Bleomycin,Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Cyclophosphamide, Cytarabine, Dacarbazine, Diethylstilbestrol, Eribulin,Ethinyl, estradiol, Etoposide, Mitomycin, Mitotane, Mitoxantrone,Pentastatin, Pipobroman, Plicamycin, Prednisone, Procarbazine,Streptozocin, Tamoxifen, Teniposide, Vinblastine, and Vincristine. 17.The selective CXCR4 binding peptide conjugate according to claim 1 ofthe formula:

wherein a, b, AA¹, X¹, AA², AA³, AA⁴, Y¹, Y², L², Z, and Ar¹ are asdefined in claim 1; and L^(1a) is a polymeric linker comprising fromabout 2 to about 20 monomers or copolymers.
 18. The selective CXCR4binding peptide conjugate according to claim 1, wherein L² is anon-polymeric linker of the formula: —CH₂—C(═O)—NH—CH₂—,—CH₂—NH—C(═O)—CH₂—, —(CH₂)_(n)—C(═O)—NH—CH₂—, —(CH₂)_(n)—NH—C(═O)—CH₂—,or —(CH₂)_(n)—, wherein n is an integer from 1 to
 6. 19. The selectiveCXCR4 binding peptide conjugate according to claim 1, wherein AA¹ alongwith the sulfur atom that is attached thereto is optionally substitutedcysteine.
 20. The selective CXCR4 binding peptide conjugate according toclaim 19, wherein α-amino group of AA¹ is substituted with an acetylgroup.
 21. The selective CXCR4 binding peptide conjugate according toclaim 1, wherein AA² along with the sulfur atom that is attached theretois optionally substituted cysteine.
 22. The selective CXCR4 bindingpeptide conjugate according to claim 1, wherein X¹ is Lys(iPr) orLys(deuterated-iPr).
 23. The selective CXCR4 binding peptide conjugateaccording to claim 1, wherein AA³ is Lys(iPr) or Lys(deuterated-iPr).24. The selective CXCR4 binding peptide conjugate according to claim 1,wherein Ar¹ is phenyl.
 25. The selective CXCR4 binding peptide conjugateaccording to claim 1, wherein b is
 1. 26. The selective CXCR4 bindingpeptide conjugate according to claim 25, wherein AA³ is Lys(iPr) orLys(deuterated-iPr).
 27. The selective CXCR4 binding peptide conjugateaccording to claim 26, wherein L¹ is a polymeric linker of the formula:—NH—(CH₂)₂—[O—CH₂—CH₂]_(n)—O—(CH₂)₂—C(═O)—, wherein n is an integer from2 to 20, —NH— is attached to the carbonyl carbon of amino acid AA³, and—C(═O)— is attached to α-amino functional group of amino acid AA⁴. 28.The selective CXCR4 binding peptide conjugate according to claim 1,wherein AA⁴ is cysteine, homocysteine, or penicillamine.
 29. Theselective CXCR4 binding peptide conjugate according to claim 1, whereinat least one of AA¹, X¹, AA², or AA³ amino acid residue is a (D)-isomer.30. The selective CXCR4 binding peptide conjugate according to claim 1,wherein said selective CXCR4 binding peptide conjugate is selected fromthe group consisting of Compound A, Compound B, Compound C, Compound D,Compound E, Compound F, Compound G, Compound H, Compound I, Compound J,Compound K, Compound L, Compound M, Compound N, Compound O, Compound P,corresponding deuterium labelled compounds thereof, and correspondingtritium labelled compounds thereof.
 31. A method for treating a subjectsuffering from a cancer, said method comprising administering to thesubject a therapeutically effective amount of a selective CXCR4 bindingpeptide conjugate of claim 1, wherein said medically useful compound isan anticancer agent.
 32. The method of claim 31, wherein said anticanceragent is selected from the group consisting of paclitaxel, Altretamine,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,Dacarbazine, Diethylstilbestrol, Eribulin, Ethinyl, estradiol,Etoposide, Mitomycin, Mitotane, Mitoxantrone, Pentastatin, Pipobroman,Plicamycin, Prednisone, Procarbazine, Streptozocin, Tamoxifen,Teniposide, Vinblastine, and Vincristine.
 33. The method of claim 31,wherein said cancer is selected from the group consisting of breastcancer, lung cancer, pancreatic cancer, kidney cancer, brain cancer,blood cancer, leukemia, prostate cancer, ovarian cancer, and bladdercancer.